Compositions of n-[2,4-bis(1,1-dimethylethyl)-5-hydroxyphenyl]-1,4-dihydro-4-oxoquinoline-3-carboxamide

ABSTRACT

Pharmaceutical compositions including N-[2,4-bis(1,1-dimethylethyl)-5-hydroxyphenyl]-1,4-dihydro-4-oxoquinoline-3-carboxamide (Compound 1) and methods of using such compositions are described herein.

This application claims priority under 35 USC §119(e) to U.S. PatentApplication Ser. No. 60/799,795, filed on May 12, 2006, the entirecontents of which are hereby incorporated by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to pharmaceutical compositions ofN-[2,4-bis(1,1-dimethylethyl)-5-hydroxyphenyl]-1,4-dihydro-4-oxoquinoline-3-carboxamideand methods therewith.

BACKGROUND OF THE INVENTION

CFTR is a cAMP/ATP-mediated anion channel that is expressed in a varietyof cells types, including absorptive and secretory epithelia cells,where it regulates anion flux across the membrane, as well as theactivity of other ion channels and proteins. In epithelia cells, normalfunctioning of CFTR is critical for the maintenance of electrolytetransport throughout the body, including respiratory and digestivetissue. CFTR is composed of approximately 1480 amino acids that encode aprotein made up of a tandem repeat of transmembrane domains, eachcontaining six transmembrane helices and a nucleotide binding domain.The two transmembrane domains are linked by a large, polar, regulatory(R)-domain with multiple phosphorylation sites that regulate channelactivity and cellular trafficking.

The gene encoding CFTR has been identified and sequenced (See Gregory,R. J. et al. (1990) Nature 347:382-386; Rich, D. P. et al. (1990) Nature347:358-362), (Riordan, J. R. et al. (1989) Science 245:1066-1073). Adefect in this gene causes mutations in CFTR resulting in cysticfibrosis (“CF”), the most common fatal genetic disease in humans. Cysticfibrosis affects approximately one in every 2,500 infants in the UnitedStates. Within the general United States population, up to 10 millionpeople carry a single copy of the defective gene without apparent illeffects. In contrast, individuals with two copies of the CF associatedgene suffer from the debilitating and fatal effects of CF, includingchronic lung disease.

In patients with cystic fibrosis, mutations in CFTR endogenouslyexpressed in respiratory epithelia leads to reduced apical anionsecretion causing an imbalance in ion and fluid transport. The resultingdecrease in anion transport contributes to enhanced mucus accumulationin the lung and the accompanying microbial infections that ultimatelycause death in CF patients. In addition to respiratory disease, CFpatients typically suffer from gastrointestinal problems and pancreaticinsufficiency that, if left untreated, results in death. In addition,the majority of males with cystic fibrosis are infertile and fertilityis decreased among females with cystic fibrosis. In contrast to thesevere effects of two copies of the CF associated gene, individuals witha single copy of the CF associated gene exhibit increased resistance tocholera and to dehydration resulting from diarrhea—perhaps explainingthe relatively high frequency of the CF gene within the population.

Sequence analysis of the CFI R gene of CF chromosomes has revealed avariety of disease causing mutations (Cutting, G. R. et al. (1990)Nature 346:366-369; Dean, M. et al. (1990) Cell 61:863:870; and Kerem,B-S. et al. (1989) Science 245:1073-1080; Kerem, B-S et al. (1990) Proc.Natl. Acad. Sci. USA 87:8447-8451). To date, >1000 disease causingmutations in the CF gene have been identified(http://www.genet.sickkids.on.ca/cftr/). The most prevalent mutation isa deletion of phenylalanine at position 508 of the CFTR amino acidsequence, and is commonly referred to as ΔF508-CFTR. This mutationoccurs in approximately 70% of the cases of cystic fibrosis and isassociated with a severe disease.

The deletion of residue 508 in ΔF508-CFTR prevents the nascent proteinfrom folding correctly. This results in the inability of the mutantprotein to exit the ER, and traffic to the plasma membrane. As a result,the number of channels present in the membrane is far less than observedin cells expressing wild-type CFTR. In addition to impaired trafficking,the mutation results in defective channel gating. Together, the reducednumber of channels in the membrane and the defective gating lead toreduced anion transport across epithelia leading to defective ion andfluid transport. (Quinton, P. M. (1990), FASEB J. 4: 2709-2727). Studieshave shown, however, that the reduced numbers of ΔF508-CFTR in themembrane are functional, albeit less than wild-type CFTR. (Dalemans etal. (1991), Nature Lond. 354: 526-528; Denning et al., supra; Pasyk andFoskett (1995), J. Cell. Biochem. 270: 12347-50). In addition toΔF508-CFTR, other disease causing mutations in CFTR that result indefective trafficking, synthesis, and/or channel gating could be up- ordown-regulated to alter anion secretion and modify disease progressionand/or severity.

Although CFTR transports a variety of molecules in addition to anions,it is clear that this role (the transport of anions) represents oneelement in an important mechanism of transporting ions and water acrossthe epithelium. The other elements include the epithelial Na⁺ channel,ENaC, Na⁺/2Cl⁻K⁺ co-transporter, Na⁺—K⁺-ATPase pump and the basolateralmembrane K⁺ channels, that are responsible for the uptake of chlorideinto the cell.

These elements work together to achieve directional transport across theepithelium via their selective expression and localization within thecell. Chloride absorption takes place by the coordinated activity ofENaC and CFTR present on the apical membrane and the Na⁺—K⁺-ATPase pumpand Cl— channels expressed on the basolateral surface of the cell.Secondary active transport of chloride from the luminal side leads tothe accumulation of intracellular chloride, which can then passivelyleave the cell via Cl⁻ channels, resulting in a vectorial transport.Arrangement of Na⁺/2Cl⁻K⁺ co-transporter, Na⁺—K⁺-ATPase pump and thebasolateral membrane K⁺ channels on the basolateral surface and CFTR onthe luminal side coordinate the secretion of chloride via CFTR on theluminal side. Because water is probably never actively transporteditself, its flow across epithelia depends on tiny transepithelialosmotic gradients generated by the bulk flow of sodium and chloride.

In addition to cystic fibrosis, modulation of CFTR activity may bebeneficial for other diseases not directly caused by mutations in CFTR,such as secretory diseases and other protein folding diseases mediatedby CFTR. These include, but are not limited to, chronic obstructivepulmonary disease (COPD), dry eye disease, and Sjögren's Syndrome. COPDis characterized by airflow limitation that is progressive and not fullyreversible. The airflow limitation is due to mucus hypersecretion,emphysema, and bronchiolitis. Activators of mutant or wild-type CFTRoffer a potential treatment of mucus hypersecretion and impairedmucociliary clearance that is common in COPD. Specifically, increasinganion secretion across CFTR may facilitate fluid transport into theairway surface liquid to hydrate the mucus and optimized periciliaryfluid viscosity. This would lead to enhanced mucociliary clearance and areduction in the symptoms associated with COPD. Dry eye disease ischaracterized by a decrease in tear aqueous production and abnormal tearfilm lipid, protein and mucin profiles. There are many causes of dryeye, some of which include age, Lasik eye surgery, arthritis,medications, chemical/thermal burns, allergies, and diseases, such ascystic fibrosis and Sjögrens's syndrome. Increasing anion secretion viaCFTR would enhance fluid transport from the corneal endothelial cellsand secretory glands surrounding the eye to increase corneal hydration.This would help to alleviate the symptoms associated with dry eyedisease. Sjögrens's syndrome is an autoimmune disease in which theimmune system attacks moisture-producing glands throughout the body,including the eye, mouth, skin, respiratory tissue, liver, vagina, andgut. Symptoms, include, dry eye, mouth, and vagina, as well as lungdisease. The disease is also associated with rheumatoid arthritis,systemic lupus, systemic sclerosis, and polymypositis/dermatomyositis.Defective protein trafficking is believed to cause the disease, forwhich treatment options are limited. Modulators of CFTR activity mayhydrate the various organs afflicted by the disease and help to elevatethe associated symptoms.

As discussed above, it is believed that the deletion of residue 508 inΔF508-CFTR prevents the nascent protein from folding correctly,resulting in the inability of this mutant protein to exit the ER, andtraffic to the plasma membrane. As a result, insufficient amounts of themature protein are present at the plasma membrane and chloride transportwithin epithelial tissues is significantly reduced. Infact, thiscellular phenomenon of defective ER processing of ABC transporters bythe ER machinery, has been shown to be the underlying basis not only forCF disease, but for a wide range of other isolated and inheriteddiseases. The two ways that the ER machinery can malfunction is eitherby loss of coupling to ER export of the proteins leading to degradation,or by the ER accumulation of these defective/misfolded proteins [AridorM, et al., Nature Med., 5(7), pp 745-751 (1999); Shastry, B. S., et al.,Neurochem. International, 43, pp 1-7 (2003); Rutishauser, J., et al.,Swiss Med Wkly, 132, pp 211-222 (2002); Morello, J P et al., TIPS, 21,pp. 466-469 (2000); Bross P., et al., Human Mut., 14, pp. 186-198(1999)]. The diseases associated with the first class of ER malfunctionare cystic fibrosis (due to misfolded ΔF508-CFTR as discussed above),hereditary emphysema (due to a1-antitrypsin; non Piz variants),hereditary hemochromatosis, hoagulation-fibrinolysis deficiencies, suchas protein C deficiency, Type 1 hereditary angioedema, lipid processingdeficiencies, such as familial hypercholesterolemia, Type 1chylomicronemia, abetalipoproteinemia, lysosomal storage diseases, suchas I-cell disease/pseudo-Hurler, Mucopolysaccharidoses (due to lysosomalprocessing enzymes), Sandhof/Tay-Sachs (due to β-hexosaminidase),Crigler-Najjar type II (due to UDP-glucuronyl-sialyc-transferase),polyendocrinopathy/hyperinsulemia, Diabetes mellitus (due to insulinreceptor), Laron dwarfism (due to growth hormone receptor),myleoperoxidase deficiency, primary hypoparathyroidism (due topreproparathyroid hormone), melanoma (due to tyrosinase). The diseasesassociated with the latter class of ER malfunction are Glycanosis CDGtype 1, hereditary emphysema (due to α1-Antitrypsin (PiZ variant),congenital hyperthyroidism, osteogenesis imperfecta (due to Type I, II,IV procollagen), hereditary hypofibrinogenemia (due to fibrinogen), ACTdeficiency (due to α1-antichymotrypsin), Diabetes insipidus (DI),neurophyseal DI (due to vasopvessin hormone/V2-receptor), neprogenic DI(due to aquaporin II), Charcot-Marie Tooth syndrome (due to peripheralmyelin protein 22), Perlizaeus-Merzbacher disease, neurodegenerativediseases such as Alzheimer's disease (due to βAPP and presenilins),Parkinson's disease, amyotrophic lateral sclerosis, progressivesupranuclear plasy, Pick's disease, several polyglutamine neurologicaldisorders asuch as Huntington, spinocerebullar ataxia type I, spinal andbulbar muscular atrophy, dentatorubal pallidoluysian, and myotonicdystrophy, as well as spongiform encephalopathies, such as hereditaryCreutzfeldt-Jakob disease (due to prion protein processing defect),Fabry disease (due to lysosomal α-galactosidase A) andStraussler-Scheinker syndrome (due to Prp processing defect).

In addition to up-regulation of CFTR activity, reducing anion secretionby CFTR modulators may be beneficial for the treatment of secretorydiarrheas, in which epithelial water transport is dramatically increasedas a result of secretagogue activated chloride transport. The mechanisminvolves elevation of cAMP and stimulation of CFTR.

Although there are numerous causes of diarrhea, the major consequencesof diarrheal diseases, resulting from excessive chloride transport arecommon to all, and include dehydration, acidosis, impaired growth anddeath.

Acute and chronic diarrheas represent a major medical problem in manyareas of the world. Diarrhea is both a significant factor inmalnutrition and the leading cause of death (5,000,000 deaths/year) inchildren less than five years old.

Secretory diarrheas are also a dangerous condition in patients ofacquired immunodeficiency syndrome (AIDS) and chronic inflammatory boweldisease (IBD). 16 million travelers to developing countries fromindustrialized nations every year develop diarrhea, with the severityand number of cases of diarrhea varying depending on the country andarea of travel.

Diarrhea in barn animals and pets such as cows, pigs and horses, sheep,goats, cats and dogs, also known as scours, is a major cause of death inthese animals. Diarrhea can result from any major transition, such asweaning or physical movement, as well as in response to a variety ofbacterial or viral infections and generally occurs within the first fewhours of the animal's life.

The most common diarrheal causing bacteria is enterotoxogenic E. coli(ETEC) having the K99 pilus antigen. Common viral causes of diarrheainclude rotavirus and coronavirus. Other infectious agents includecryptosporidium, giardia lamblia, and salmonella, among others.

Symptoms of rotaviral infection include excretion of watery feces,dehydration and weakness. Coronavirus causes a more severe illness inthe newborn animals, and has a higher mortality rate than rotaviralinfection. Often, however, a young animal may be infected with more thanone virus or with a combination of viral and bacterial microorganisms atone time. This dramatically increases the severity of the disease.

Accordingly, there is a need for pharmaceutical compositions ofmodulators of CFTR activity that can be used to modulate the activity ofCFTR in the cell membrane of a mammal.

There is a need for methods of treating CFTR-mediated diseases usingsuch pharmaceutical compositions.

SUMMARY OF THE INVENTION

The present invention relates to pharmaceutical compositions ofN-[2,4-bis(1,1-dimethylethyl)-5-hydroxyphenyl]-1,4-dihydro-4-oxoquinoline-3-carboxamide(hereinafter “Compound 1”) which has the structure below:

The pharmaceutical compositions of Compound 1 are useful for treating orlessening the severity of a variety of CFTR-mediated diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an X-Ray powder diffraction pattern of Compound 1.

FIG. 2 is the ¹H NMR spectrum of Compound 1.

FIG. 3 is the DSC trace of Compound 1.

DETAILED DESCRIPTION OF THE INVENTION

According to one embodiment, the present invention provides apharmaceutical composition comprising:

(i)N-[2,4-bis(1,1-dimethylethyl)-5-hydroxyphenyl]-1,4-dihydro-4-oxoquinoline-3-carboxamide(Compound 1) or a pharmaceutically acceptable salt thereof;

(ii) a suitable liquid PEG; and

(iii) optionally, a suitable viscosity enhancing agent.

As used herein, the phrase “suitable liquid PEG” means a polyethyleneglycol polymer that is in liquid form at ambient temperature and isamenable for use in a pharmaceutical composition. Such suitablepolyethylene glycols are well known in the art; see, e.g.,http://www.medicinescomelete.com/mc/excipients/current, which isincorporated herein by reference. Exemplary PEGs include low molecularweight PEGs such as PEG 200, PEG 300, PEG 400, etc. The number thatfollows the term “PEG” indicates the average molecular weight of thatparticular polymer. E.g., PEG 400 is a polyethylene glycol polymerwherein the average molecular weight of the polymer therein is about400.

In one embodiment, said suitable liquid PEG has an average molecularweight of from about 200 to about 600. In another embodiment, saidsuitable liquid PEG is PEG 400 (for example a PEG having a molecularweight of form about 380 to about 420 g/mol).

In another embodiment, the present invention provides a pharmaceuticalcomposition comprising Compound 1 or a pharmaceutically acceptable saltthereof; propylene glycol; and, optionally, a suitable viscosityenhancing agent.

In another embodiment, the pharmaceutical compositions of the presentinvention comprise a suitable viscosity enhancing agent. In oneembodiment, the suitable viscosity enhancing agent is a polymer solublein PEG. Such suitable viscosity enhancing agents are well known in theart, e.g., polyvinyl pyrrolidine (hereinafter “PVP”). PVP ischaracterized by its viscosity in aqueous solution, relative to that ofwater, expressed as a K-value (denoted as a suffix, e.g., PVP K20), inthe range of from about 10 to about 120. See, e.g.,http://www.medicinescomplete.com/mc/excipients/current. Embodiments ofPVP useful in the present invention have a K-value of about 90 or less.An exemplary such embodiment is PVP K30.

In one embodiment, the present invention provides a pharmaceuticalcomposition comprising:

-   -   (i)        N-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide        (Compound 1) or a pharmaceutically acceptable salt thereof;    -   (ii) PEG 400; and    -   (iii) PVP K30.

In another embodiment, the present invention provides a pharmaceuticalcomposition, wherein saidN-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamideis present in an amount from about 0.01% w/w to about 6.5 w/w.

In another embodiment, the present invention provides a pharmaceuticalcomposition, wherein said PEG is present in an amount from about 87.5%w/w to about 99.99% w/w.

In another embodiment, the present invention provides a pharmaceuticalcomposition, wherein said PVP K30 is present in an amount between 0% w/wto about 6% w/w.

In another embodiment, the present invention provides a pharmaceuticalcomposition, wherein said composition comprises PEG 400 (e.g., fromabout 97.8 to about 98.0% w/w, for example, about 97.88% w/w), PVP K30(e.g., from about 1.9 to about 2.1% w/w, for example, about 2.0% w/w),and Compound 1 (e.g., from about 0.10 to about 0.15% w/w, for example,about 0.13% w/w).

In another embodiment, the present invention provides a pharmaceuticalcomposition, wherein said composition comprises PEG 400 (e.g., fromabout 97.5 to about 98.0% w/w, for example, about 97.75% w/w), PVP K30(e.g., from about 1.8 to about 2.2% w/w, for example, about 2.0% w/w),and Compound 1 (e.g., from about 0.2 to about 0.3% w/w, for example,about 0.25% w/w).

In another embodiment, the present invention provides a pharmaceuticalcomposition, wherein said composition comprises PEG 400 (e.g., fromabout 97.2 to about 97.8, for example, about 97.50% w/w), PVP K30 (e.g.,from about 1.8 to about 2.2% w/w, for example, about 2.0% w/w), andCompound 1 (e.g., from about 0.4 to about 0.6% w/w, for example, about0.50% w/w).

In another embodiment, the present invention provides a pharmaceuticalcomposition, wherein said composition comprises PEG 400 (e.g., fromabout 96.5 to about 97.5% w/w, for example, about 97.0% w/w), PVP K30(e.g., from about 1.8 to about 2.2% w/w, for example, about 2.0% w/w),and Compound 1 (e.g., from about 0.9 to about 1.1% w/w, for example,about 1.0% w/w).

In another embodiment, the present invention provides a pharmaceuticalcomposition, wherein said composition comprises PEG 400 (e.g., fromabout 96.60 to about 96.65% w/w, for example, about 96.63% w/w), PVP K30(e.g., from about 1.8 to about 2.2% w/w, for example, about 2.0% w/w),and Compound 1 (e.g., from about 1.30 to about 1.45% w/w, for example,about 1.38% w/w).

In another embodiment, the present invention provides a pharmaceuticalcomposition, wherein said composition comprises PEG 400 (e.g., fromabout 96.0 to about 96.3% w/w, for example, about 96.12% w/w), PVP K30(e.g., from about 1.8 to about 2.0% w/w, for example, about 2.0% w/w),and Compound 1 (e.g., from about 1.8 to about 2.2% w/w, for example,about 1.88% w/w).

In another embodiment, the present invention provides a pharmaceuticalcomposition, wherein said composition comprises PEG 400 (e.g., fromabout 95.5 to about 96.0% w/w, for example, about 95.75% w/w), PVP K30(e.g., from about 1.8 to about 2.2% w/w, for example, about 2.0% w/w),and Compound 1 (e.g., from about 2.0 to about 2.5% w/w, for example,about 2.25% w/w).

In another embodiment, the present invention provides a pharmaceuticalcomposition, wherein said composition comprises PEG 400 (e.g., fromabout 95 to about 96% w/w, for example, about 95.5% w/w), PVP K30 (e.g.,from about 1.8 to about 2.2% w/w, for example, about 2.0% w/w), andCompound 1 (e.g., from about 2.3 to about 2.7% w/w, for example, about2.50% w/w)

In another embodiment, the present invention provides a pharmaceuticalcomposition, wherein said composition comprises PEG 400 (e.g., fromabout 94.5 to about 94.8, for example, about 94.63% w/w), PVP K30 (e.g.,from about 1.8 to about 2.2% w/w, for example, about 2.0% w/w), andCompound 1 (e.g., from about 3.5 to about 4.0% w/w, for example, about3.38% w/w).

In another embodiment, the present invention provides a pharmaceuticalcomposition, wherein said composition comprises PEG 400 (e.g., fromabout 93.5 to about 94.5% w/w, for example, about 94.0% w/w), PVP K30(e.g., from about 1.8 to about 2.2% w/w, for example, about 2.0% w/w),and Compound 1 (e.g., from about 3.7 to about 4.3% w/w, for example,about 4.0% w/w).

In one embodiment, the present invention provides a pharmaceuticalcomposition comprising:

-   -   (i)        N-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide        (Compound 1) or a pharmaceutically acceptable salt thereof;    -   (ii) a suitable PEG lipid; and    -   (iii) PVP.

In some embodiments, the PEG lipid has an average molecular weight offrom about 400 to about 600, for example, PEG 400. In some embodiments,the PVP is PVP K30.

According to another embodiment, the pharmaceutical compositions of thepresent invention comprise a therapeutically effective amount ofCompound 1. The phrase “therapeutically effective amount” is that amounteffective for treating or lessening the severity of any of the diseases,conditions, or disorders recited below.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, (Z) and (E) double bondisomers, and (Z) and (E) conformational isomers. Therefore, singlestereochemical isomers as well as enantiomeric, diastereomeric, andgeometric (or conformational) mixtures of the present compounds arewithin the scope of the invention. Unless otherwise stated, alltautomeric forms of the compounds of the invention are within the scopeof the invention. E.g., Compound 1 may exist as tautomers:

Unless otherwise stated, structures depicted herein are also meant toinclude compounds that differ only in the presence of one or moreisotopically enriched atoms. For example, compounds of formula (I),wherein one or more hydrogen atoms are replaced deuterium or tritium, orone or more carbon atoms are replaced by a 13C- or 14C-enriched carbonare within the scope of this invention. Such compounds are useful, forexample, as analytical tools, probes in biological assays, or compoundswith improved therapeutic profile.

Uses, Formulation and Administration

Pharmaceutically Acceptable Compositions

In another aspect of the present invention, pharmaceutically acceptablecompositions are provided, wherein these compositions comprise anadditional pharmaceutically acceptable carrier, adjuvant or vehicle. Incertain embodiments, these compositions optionally further comprise oneor more additional therapeutic agents.

It will also be appreciated that certain of the compounds of presentinvention can exist in free form for treatment, or where appropriate, asa pharmaceutically acceptable derivative or a prodrug thereof. Accordingto the present invention, a pharmaceutically acceptable derivative or aprodrug includes, but is not limited to, pharmaceutically acceptablesalts, esters, salts of such esters, or any other adduct or derivativewhich upon administration to a patient in need thereof is capable ofproviding, directly or indirectly, a compound as otherwise describedherein, or a metabolite or residue thereof.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgement,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. A“pharmaceutically acceptable salt” means any non-toxic salt or salt ofan ester of a compound of this invention that, upon administration to arecipient, is capable of providing, either directly or indirectly, acompound of this invention or an inhibitorily active metabolite orresidue thereof.

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from suitable inorganic and organic acids andbases. Examples of pharmaceutically acceptable, nontoxic acid additionsalts are salts of an amino group formed with inorganic acids such ashydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid andperchloric acid or with organic acids such as acetic acid, oxalic acid,maleic acid, tartaric acid, citric acid, succinic acid or malonic acidor by using other methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄alcyl)₄ salts. This inventionalso envisions the quaternization of any basic nitrogen-containinggroups of the compounds disclosed herein. Water or oil-soluble ordispersable products may be obtained by such quaternization.Representative alkali or alkaline earth metal salts include sodium,lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, loweralkyl sulfonate and aryl sulfonate.

In one embodiment, the present invention provides a method of treating aCFTR mediated disease, condition, or disorder in a patient comprisingthe step of administering to a patient a pharmaceutical compositionaccording to the present invention.

A “CFTR-mediated disease” as used herein is a disease selected fromcystic fibrosis, Hereditary emphysema, Hereditary hemochromatosis,Coagulation-Fibrinolysis deficiencies, such as Protein C deficiency,Type 1 hereditary angioedema, Lipid processing deficiencies, such asFamilial hypercholesterolemia, Type 1 chylomicronemia,Abetalipoproteinemia, Lysosomal storage diseases, such as I-celldisease/Pseudo-Hurler, Mucopolysaccharidoses, Sandhof/Tay-Sachs,Crigler-Najjar type Polyendocrinopathy/Hyperinsulemia, Diabetesmellitus, Laron dwarfism, Myleoperoxidase deficiency, Primaryhypoparathyroidism, Melanoma, Glycanosis CDG type 1, Hereditaryemphysema, Congenital hyperthyroidism, Osteogenesis imperfecta,Hereditary hypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI),Neurophyseal DI, Neprogenic DI, Charcot-Marie Tooth syndrome,Perlizaeus-Merzbacher disease, neurodegenerative diseases such asAlzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis,Progressive supranuclear plasy, Pick's disease, several polyglutamineneurological disorders asuch as Huntington, Spinocerebullar ataxia typeI, Spinal and bulbar muscular atrophy, Dentatorubal pallidoluysian, andMyotonic dystrophy, as well as Spongiform encephalopathies, such asHereditary Creutzfeldt-Jakob disease, Fabry disease,Straussler-Scheinker syndrome, COPD, dry-eye disease, and Sjogren'sdisease.

According to an alternative embodiment, the present invention provides amethod of treating cystic fibrosis comprising the step of administeringto said mammal a pharmaceutical composition according to the presentinvention.

In certain embodiments, the pharmaceutically compositions of the presentinvention are useful for treating or lessening the severity of cysticfibrosis in patients who exhibit residual CFTR activity in the apicalmembrane of respiratory and non-respiratory epithelia. The presence ofresidual CFTR activity at the epithelial surface can be readily detectedusing methods known in the art, e.g., standard electrophysiological,biochemical, or histochemical techniques. Such methods identify CFTRactivity using in vivo or ex vivo electrophysiological techniques,measurement of sweat or salivary Cl⁻ concentrations, or ex vivobiochemical or histochemical techniques to monitor cell surface density.Using such methods, residual CFTR activity can be readily detected inpatients heterozygous or homozygous for a variety of differentmutations, including patients homozygous or heterozygous for the mostcommon mutation, ΔF508.

In one embodiment, the pharmaceutically acceptable compositions of thepresent invention are useful for treating or lessening the severity ofcystic fibrosis in patients within certain genotypes exhibiting residualCFTR activity, e.g., class III mutations (impaired regulation orgating), class IV mutations (altered conductance), or class V mutations(reduced synthesis) (Lee R. Choo-Kang, Pamela L., Zeitlin, Type I, II,III, IV, and V cystic fibrosis Tansmembrane Conductance RegulatorDefects and Opportunities of Therapy; Current Opinion in PulmonaryMedicine 6:521-529, 2000). Other patient genotypes that exhibit residualCFTR activity include patients homozygous for one of these classes orheterozygous with any other class of mutations, including class Imutations, class II mutations, or a mutation that lacks classification.

In one embodiment, the pharmaceutically acceptable composition of thepresent invention are useful for treating or lessening the severity ofcystic fibrosis in patients within certain clinical phenotypes, e.g., amoderate to mild clinical phenotype that typically correlates with theamount of residual CFTR activity in the apical membrane of epithelia.Such phenotypes include patients exhibiting pancreatic sufficiency orpatients diagnosed with idiopathic pancreatitis and congenital bilateralabsence of the vas deferens, or mild lung disease.

The exact amount of Compound 1 required in the pharmaceuticalcompositions of the present invention will vary from subject to subject,depending on the species, age, and general condition of the subject, theseverity of the infection, the particular agent, its mode ofadministration, and the like. The compounds of the invention arepreferably formulated in dosage unit form for ease of administration anduniformity of dosage. The expression “dosage unit form” as used hereinrefers to a physically discrete unit of agent appropriate for thepatient to be treated. It will be understood, however, that the totaldaily usage of the compounds and compositions of the present inventionwill be decided by the attending physician within the scope of soundmedical judgment. The specific effective dose level for any particularpatient or organism will depend upon a variety of factors including thedisorder being treated and the severity of the disorder; the activity ofthe specific compound employed; the specific composition employed; theage, body weight, general health, sex and diet of the patient; the timeof administration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed, andlike factors well known in the medical arts. The term “patient”, as usedherein, means an animal, preferably a mammal, and most preferably ahuman.

The pharmaceutically acceptable compositions of this invention can beadministered orally at dosage levels of about 0.01 mg/kg to about 50mg/kg and preferably from about 1 mg/kg to about 25 mg/kg, of subjectbody weight per day, one or more times a day, to obtain the desiredtherapeutic effect.

The pharmaceutical compositions of the present invention mayadditionally contain inert diluents commonly used in the art such as,for example, water or other solvents, solubilizing agents andemulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed,groundnut, corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

It will also be appreciated that the pharmaceutically compositions ofthe present invention can be employed in combination therapies, that is,they can be administered concurrently with, prior to, or subsequent to,one or more other desired therapeutics or medical procedures. Theparticular combination of therapies (therapeutics or procedures) toemploy in a combination regimen will take into account compatibility ofthe desired therapeutics and/or procedures and the desired therapeuticeffect to be achieved. It will also be appreciated that the therapiesemployed may achieve a desired effect for the same disorder (forexample, a pharmaceutical composition of the present invention may beadministered concurrently with another agent used to treat the samedisorder), or they may achieve different effects (e.g., control of anyadverse effects). As used herein, additional therapeutic agents normallyadministered to treat or prevent a particular disease, or condition, areknown as “appropriate for the disease, or condition, being treated”.

In one embodiment, the additional agent is selected from a mucolyticagent, bronchodialator, an anti-biotic, an anti-infective agent, ananti-inflammatory agent, a CFTR modulator other than a compound of thepresent invention, or a nutritional agent.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. Preferably the amount of additional therapeutic agentin the presently disclosed compositions will range from about 50% to100% of the amount normally present in a composition comprising thatagent as the only therapeutically active agent.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

EXAMPLES Methods & Materials Differential Scanning Calorimetry (DSC)

DSC data was collected on a TA instrument Q1000 equipped with a 50position autosampler. The energy and temperature calibration standardwas indium. Samples were heated at a rate of 10° C./min between 20 and350° C. A nitrogen purge at 30 ml/min was maintained over the sample.

Between 0.5 and 4 mg of sample was used and all samples run in a pinholealuminium pan.

NMR

All spectra were collected on a Bruker 400 MHz equipped withautosampler. Samples were prepared in d₆-DMSO, unless otherwise stated.

XRPD (X-ray Powder Diffraction)

Bruker AXS C2 GADDS Diffractometer

X-ray powder diffraction patterns for the samples were acquired on aBruker AXS C2 GADDS diffractometer using Cu Kα radiation (40 kV, 40 mA),automated XYZ stage, laser video microscope for auto-sample positioningand a HiStar 2-dimensional area detector. X-ray optics consists of asingle Göbel multilayer mirror coupled with a pinhole collimator of 0.3mm.

Beam divergence, i.e. the effective size of the X-ray beam on thesample, was approximately 4 mm. A θ-θ continuous scan mode was employedwith a sample to detector distance of 20 cm which gives an effective 2θrange of 3.2-29.8°. A typical exposure time of a sample would be 120s.

Samples run under ambient conditions were prepared as flat platespecimens using powder as received without grinding. Approximately 1-2mg of the sample was lightly pressed on a glass slide to obtain a flatsurface. Samples run under non-ambient conditions were mounted on asilicon wafer with heat conducting compound. The sample was then heatedto the appropriate temperature at ca. 20° C./minute and subsequentlyheld isothermally for ca 1 minute before data collection was initiated.

Synthesis ofN-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide(Compound 1)

2-Phenylaminomethylene-malonic acid diethyl ester

A mixture of aniline (25.6 g, 0.275 mol) and diethyl2-(ethoxymethylene)malonate (62.4 g, 0.288 mol) was heated at 140-150°C. for 2 h. The mixture was cooled to room temperature and dried underreduced pressure to afford 2-phenylaminomethylene-malonic acid diethylester as a solid, which was used in the next step without furtherpurification. ¹H NMR (DMSO-d₆) δ 11.00 (d, 1H), 8.54 (d, J=13.6 Hz, 1H),7.36-7.39 (m, 2H), 7.13-7.17 (m, 3H), 4.17-4.33 (m, 4H), 1.18-1.40 (m,6H).

4-Hydroxyquinoline-3-carboxylic acid ethyl ester

A 1 L three-necked flask fitted with a mechanical stirrer was chargedwith 2-phenylaminomethylene-malonic acid diethyl ester (26.3 g, 0.100mol), polyphosphoric acid (270 g) and phosphoryl chloride (750 g). Themixture was heated to 70° C. and stirred for 4 h. The mixture was cooledto room temperature and filtered. The residue was treated with aqueousNa₂CO₃ solution, filtered, washed with water and dried.4-Hydroxyquinoline-3-carboxylic acid ethyl ester was obtained as a palebrown solid (15.2 g, 70%). The crude product was used in next stepwithout further purification.

4-Oxo-1,4-dihydroquinoline-3-carboxylic acid

4-Hydroxyquinoline-3-carboxylic acid ethyl ester (15 g, 69 mmol) wassuspended in sodium hydroxide solution (2N, 150 mL) and stirred for 2 hat reflux. After cooling, the mixture was filtered, and the filtrate wasacidified to pH 4 with 2N HCl. The resulting precipitate was collectedvia filtration, washed with water and dried under vacuum to give4-oxo-1,4-dihydroquinoline-3-carboxylic acid as a pale white solid (10.5g, 92%). ¹H NMR (DMSO-d₆) δ 15.34 (s, 1H), 13.42 (s, 1H), 8.89 (s, 1H),8.28 (d, J=8.0 Hz, 1H), 7.88 (m, 1H), 7.81 (d, J=8.4 Hz, 1H), 7.60 (m,1H).

Carbonic acid 2,4-di-tert-butyl-phenyl ester methyl ester

Methyl chloroformate (58 mL, 750 mmol) was added dropwise to a solutionof 2,4-di-ten-butyl-phenol (103.2 g, 500 mmol), Et₃N (139 mL, 1000 mmol)and DMAP (3.05 g, 25 mmol) in dichloromethane (400 mL) cooled in anice-water bath to 0° C. The mixture was allowed to warm to roomtemperature while stirring overnight, then filtered through silica gel(approx. 1 L) using 10% ethyl acetate-hexanes (˜4 L) as the eluent. Thecombined filtrates were concentrated to yield carbonic acid2,4-di-tert-butyl-phenyl ester methyl ester as a yellow oil (132 g,quant.). NMR (400 MHz, DMSO-d₆) δ 7.35 (d, J=2.4 Hz, 1H), 7.29 (dd,J=8.5, 2.4 Hz, 1H), 7.06 (d, J=8.4 Hz, 1H), 3.85 (s, 3H), 1.30 (s, 9H),1.29 (s, 9H).

Carbonic acid 2,4-di-tert-butyl-5-nitro-phenyl ester methyl ester andCarbonic acid 2,4-di-tert-butyl-6-nitro-phenyl ester methyl ester

To a stirring mixture of carbonic acid 2,4-di-tert-butyl-phenyl estermethyl ester (4.76 g, 180 mmol) in conc. sulfuric acid (2 mL), cooled inan ice-water bath, was added a cooled mixture of sulfuric acid (2 mL)and nitric acid (2 mL). The addition was done slowly so that thereaction temperature did not exceed 50° C. The reaction was allowed tostir for 2 h while warming to room temperature. The reaction mixture wasthen added to ice-water and extracted into diethyl ether. The etherlayer was dried (MgSO₄), concentrated and purified by columnchromatography (0-10% ethyl acetate-hexanes) to yield a mixture ofcarbonic acid 2,4-di-ten-butyl-5-nitro-phenyl ester methyl ester andcarbonic acid 2,4-di-ten-butyl-6-nitro-phenyl ester methyl ester as apale yellow solid (4.28 g), which was used directly in the next step.

2,4-Di-tert-butyl-5-nitro-phenol and 2,4-DI-tert-butyl-6-nitro-phenol

The mixture of carbonic acid 2,4-di-tert-butyl-5-nitro-phenyl estermethyl ester and carbonic acid 2,4-di-ten-butyl-6-nitro-phenyl estermethyl ester (4.2 g, 14.0 mmol) was dissolved in MeOH (65 mL) before KOH(2.0 g, 36 mmol) was added. The mixture was stirred at room temperaturefor 2 h. The reaction mixture was then made acidic (pH 2-3) by addingconc. HCl and partitioned between water and diethyl ether. The etherlayer was dried (MgSO₄), concentrated and purified by columnchromatography (0-5% ethyl acetate-hexanes) to provide2,4-di-tert-butyl-5-nitro-phenol (1.31 g, 29% over 2 steps) and2,4-di-tert-butyl-6-nitro-phenol. 2,4-Di-tert-butyl-5-nitro-phenol: ¹HNMR (400 MHz, DMSO-d₆) δ 10.14 (s, 1H, OH), 7.34 (s, 1H), 6.83 (s, 1H),1.36 (s, 9H), 1.30 (s, 9H). 2,4-Di-ten-butyl-6-nitro-phenol: ¹H NMR (400MHz, CDCl₃) δ 11.48 (s, 1H), 7.98 (d, J=2.5 Hz, 1H), 7.66 (d, J=2.4 Hz,1H), 1.47 (s, 9H), 1.34 (s, 9H).

5-Amino-2,4-di-tert-butyl-phenol

To a reluxing solution of 2,4-di-ten-butyl-5-nitro-phenol (1.86 g, 7.40mmol) and ammonium formate (1.86 g) in ethanol (75 mL) was added Pd-5%wt. on activated carbon (900 mg). The reaction mixture was stirred atreflux for 2 h, cooled to room temperature and filtered through Celite.The Celite was washed with methanol and the combined filtrates wereconcentrated to yield 5-amino-2,4-di-ten-butyl-phenol as a grey solid(1.66 g, quant.). ¹H NMR (400 MHz, DMSO-d₆) δ 8.64 (s, 1H, OH), 6.84 (s,1H), 6.08 (s, 1H), 4.39 (s, 2H, NH₂), 1.27 (m, 18H); HPLC ret. time 2.72min, 10-99% CH₃CN, 5 min run; ESI-MS 222.4 m/z [M+H]⁺.

N-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide

To a suspension of 4-oxo-1,4-dihydroquinolin-3-carboxylic acid (35.5 g,188 mmol) and HBTU (85.7 g, 226 mmol) in DMF (280 mL) was added Et₃N(63.0 mL, 451 mmol) at ambient temperature. The mixture becamehomogeneous and was allowed to stir for 10 min before5-amino-2,4-di-tert-butyl-phenol (50.0 g, 226 mmol) was added in smallportions. The mixture was allowed to stir overnight at ambienttemperature. The mixture became heterogeneous over the course of thereaction. After all of the acid was consumed (LC-MS analysis, MH+ 190,1.71 min), the solvent was removed in vacuo. EtOH was added to theorange solid material to produce a slurry. The mixture was stirred on arotovap (bath temperature 65° C.) for 15 min without placing the systemunder vacuum. The mixture was filtered and the captured solid was washedwith hexanes to provide a white solid that was the EtOH crystalate. Et₂Owas added to the material obtained above until a slurry was formed. Themixture was stirred on a rotovapor (bath temperature 25° C.) for 15 minwithout placing the system under vacuum. The mixture was filtered andthe solid captured. This procedure was performed a total of five times.The solid obtained after the fifth precipitation was placed under vacuumovernight to provide 8N-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamideas a white powdery solid (38 g, 52%).

HPLC ret. time 3.45 min, 10-99% CH₃CN, 5 min run; ¹H NMR (400 MHz,DMSO-d₆) δ 12.88 (s, 1H), 11.83 (s, 1H), 9.20 (s, 1H), 8.87 (s, 1H),8.33 (dd, J=8.2, 1.0 Hz, 1H), 7.83-7.79 (m, 1H), 7.76 (d, J=7.7 Hz, 1H),7.54-7.50 (m, 1H), 7.17 (s, 1H), 7.10 (s, 1H), 1.38 (s, 9H), 1.37 (s,9H); ESI-MS 393.3 m/z [M+H]⁺.

Set forth below is the characterizing data for Compound 1:

TABLE 2 Cmd LC-MS LC-RT No. M + 1 min 1 393.2 3.71

The XRPD spectrum of Compound 1 is shown in FIG. 1.

¹H NMR data for Compound 1 in shown in FIG. 2.

The DSC trace of Compound 1 is shown in FIG. 3.

Preparation of Pharmaceutical Compositions Materials

A Glass bottle for formulation preparation (250 cc amber glass withteflon lined lid)

Glass bottle for dose confirmation sample (30 cc amber glass with Teflonlined lid)

Stir Plate with temperature probe (ensure probe has been cleaned)

New magnetic stir bar

Spatulas for dispensing excipient and active.

Step 1: To a clean 250 cc amber glass bottle add the stir bar to thebottle and record the tare weight of the bottle, stir bar, label andcap. Tare the bottle with the label and stir bar.

Step 2: Dispense targeted amount of PEG400 into the bottle andaccurately weigh. Place the bottle on stir plate and stir to form asmall vortex at the surface of the liquid (˜300-500 rpm or asnecessary). Insert the cleaned temperature probe into the liquid to adepth of ˜1 cm and raise the setpoint of the heater to 40° C. Cover thebottle opening with aluminum foil. Allow the PEG400 to stabilize at40+/−5° C.

Step 3: Dispense the required amount of PVP K30 and add to the stirringPEG400. Add the PVP in a slow stream (over ˜2-3 minutes) and allow theparticles to disperse. If the particles clump, the dissolution will takelonger. Cover the bottle opening with foil and continue stirring themixture at 40+/−5° C. The mixture should be sampled at 10 minutes usinga small transfer pipette to determine if the PVP has completelydissolved. The stirring solution should also be examined for large,undissolved clumps. If the solution is clear, proceed to the next step.If undissolved polymer remains, continue stirring. Check for dissolutionevery 10 minutes, with a maximum stirring time of 30 minutes total. Whencomplete dissolution is observed, proceed to the next step. If completedissolution is not observed within 30 minutes after PVP addition,terminate preparation, discard the material, and start the preparationfrom the beginning.

Step 4: Dispense the required amount of Compound 1 and add to thestirred PEG/PVP solution in a slow stream. Cover the bottle opening withfoil and continue stirring the mixture at 40+/−5° C. The mixture shouldbe sampled after 30 minutes using a small transfer pipette to determineif the Compound 1 has completely dissolved. If the solution is clearafter 30 minutes, proceed to the next step. If undissolved Compound 1remains, continue stirring. Check for dissolution every 30 minutes witha maximum stirring time of 300 minutes (5 hours) after addition ofCompound 1. If complete dissolution is not observed within 300 minutes(5 hours) after addition of Compound 1, terminate preparation, discardthe material, and start the preparation from the beginning.

Upon complete dissolution of the Compound 1, remove from the stir plate,and cap the bottle. The formulation should be maintained at roomtemperature until dosing, but must be dosed within 24 hours ofpreparation. If precipitation of VX-770 is observed, do not dose thesolution.

Using the above method, the following ten pharmaceutical compositions inTable A were prepared:

TABLE A % PEG % PVP % Cmpd 1 Amount of Cmpd 1 Composition # 400 w/w K30w/w w/w per 20 g dose (mg) 1 97.875 2.0 0.125 25 2 97.750 2.0 0.250 50 397.500 2.0 0.500 100 4 97.000 2.0 1.000 200 5 96.625 2.0 1.375 275 696.125 2.0 1.875 375 7 95.750 2.0 2.25 450 8 95.500 2.0 2.500 500 994.625 2.0 3.375 675 10 94.000 2.0 4.000 800

1. A pharmaceutical composition comprising: (i)N-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamideor a pharmaceutically acceptable salt thereof; (ii) a liquid PEG whichhas an average molecular weight of between about 200 and about 600; and(iii) optionally, PVP.
 2. (canceled)
 3. The pharmaceutical compositionaccording to claim 1, wherein said PVP has a K-value of about 30 orless.
 4. The pharmaceutical composition according to claim 3, whereinsaid PVP has a K-value of
 30. 5-24. (canceled)